Integrated system and methods for real-time anatomical guidance in a diagnostic or therapeutic procedure

ABSTRACT

According to one aspect, a system for intraoperatively providing anatomical guidance in a diagnostic or therapeutic procedure is disclosed. In one embodiment, the system includes: a first light source configured to emit a beam of visible light; second light source configured to emit a beam of near-infrared light; a handheld probe optically coupled to the second light source; a second imaging device configured to detect visible light; a third imaging device configured to detect near-infrared light having a first predetermined wavelength; a fourth imaging device configured to detect near-infrared light having a second predetermined wavelength; a display for displaying at least one visual representation of data; and, a controller programmed to generate at least one real-time integrated visual representation of an area of interest and to display the real-time visual representation on the display for guidance during the diagnostic or therapeutic procedure.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 12/969,396, filed Dec. 15, 2010 and published Jun.23, 2011 as U.S. Patent Appl. Publication No. 2011/0152692, nowabandoned, which claims the benefit, pursuant to 35 U.S.C. §119(e), ofprovisional U.S. Patent Application Ser. No. 61/286,519, filed Dec. 15,2009 and now expired, entitled “SYSTEM AND METHODS FOR INTRAOPERATIVELYPROVIDING ANATOMICAL GUIDANCE IN A SURICAL PROCEDURE,” by Shuming Nie,Aaron Mohs, and Michael Mancini, and provisional U.S. Patent ApplicationSer. No. 61/385,613, filed Sep. 23, 2010 and now expired, entitled “AHANDHELD SPECTROSCOPIC DEVICE FOR IN VIVO AND INTRA-OPERATIVE TUMORDETECTION: CONTRAST ENHANCEMENT, DETECTION SENSITIVITY, AND TISSUEPENETRATION,” by Shuming Nie, Aaron Mohs, and Michael Mancini, thedisclosures of which are herein incorporated by reference in theirentireties.

STATEMENT OF FEDERALLY-SPONSORED RESEARCH

This invention was made with Government support under Grant No.U54CA011933 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

Some references, which may include patents, patent applications andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference. In terms of notation, hereinafter, “[n]”represents the nth reference cited in the reference list. For example,[4] represents the 4^(th) reference cited in the reference list, namely,Karakiewicz, P. I. et al., Urology 2005, 66, 1245-1250.

FIELD OF THE INVENTION

The present invention generally relates to systems and methods forintraoperatively providing guidance in a diagnostic or therapeuticprocedure.

BACKGROUND OF THE INVENTION

Many forms of human cancer are treated by surgical resection,chemotherapy, and/or radiation. Surgery provides significant survivaladvantages for a broad range of tumor types and cures approximately 45%of all patients with solid tumors [1]. To successfully treat a patientwith surgery, the surgeon must remove the entire tumor at the time ofsurgery including the primary tumor, draining lymph nodes that maycontain tumor cells and small adjacent satellite nodules. Statisticaldata indicate that complete resection is the single most importantpredictor of patient survival for almost all solid tumors [2]. In lung,breast, prostate, colon, and pancreatic cancers, a complete resectionhas a 3-5 fold improvement in survival as compared to partial resection[3-8]. Recent advances in computed tomography (CT), positron emissiontomography (PET), and hybrid techniques (such as CT/PET) have greatlyimproved tumor detection and surgical planning [9,10], but thesemodalities do not provide real-time intra-operative assistance.Intra-operative magnetic resonance imaging (MRI) can assist in surgicalresection of tumors, but it is time consuming and substantially adds tothe length of surgery, anesthesia time, and financial costs [11].Intra-operative sonography has also shown potential for detection ofbreast cancer but has limited sensitivity for detection of masses lessthan 5 mm [12]. Faced with these difficulties, optical technologiesbased on cellular imaging, native fluorescence, and Raman scatteringhave gained attention for tumor detection and diagnosis [13-17]. Inparticular, the level of autofluorescence from collagen, nicotinamideadenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) hasbeen associated with malignancy in head and neck cancer [17-19].Chemical and biochemical changes have been measured by laser Ramanspectroscopy for margin assessment of breast cancer [15, 20] and fornoninvasive detection of cervical dysplasia during routine pelvic exams[21]. Small changes in cellular biochemistry may translate intospectroscopic differences that are measurable with fluorescence or Ramanscattering. However, tumors are highly heterogeneous in their molecularand cellular compositions [22], and biochemical differences in malignantand benign tissues are subject to natural variations in patientphysiology and pathology [23]. Thus, autofluorescence and intrinsicRaman measurements often lead to unacceptable false-positive rates forbenign tissues and unacceptable false-negative rates for malignanttissues [24, 25].

Due to tissue scattering and blood absorption, optical methods haverelatively limited penetration depths [26, 27]. For intra-operativeapplications, however, the lesions are surgically exposed and can bebrought in close proximity to the imaging device, so they becomeaccessible to optical illumination and detection. A problem in usingexogenous contrast agents is that they are often unable to deeplypenetrate solid tumors, especially when macromolecules such asmonoclonal antibodies or nanoparticles are used [28-30]. For detectionof tumor margins during surgery, on the other hand, the agents aredetected at the tumor periphery and deep penetration is not required.Similarly, for detection of small and residual tumors, deep penetrationis not required because small tumors do not have a high intra-tumoralpressure or a necrotic/hypoxic core, two factors in limiting tumorpenetration of imaging and therapeutic agents [28-30].

There is a need for anatomical guidance and rapid pathology to beprovided during the diagnostic or therapeutic procedure, to determine ifa tumor has been completely resected, such as by verifying that themargin of resected tumor tissue is clear, without having to wait forpathology to process the resected tissue to verify that there are noremaining signs of cancerous growth in the margin.

Therefore, a heretofore unaddressed need still exists in the art toaddress the aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a system forintraoperatively providing anatomical guidance in a diagnostic ortherapeutic procedure. In one embodiment, the system includes a firstlight source that is configured to emit a beam of visible light to anarea of interest of a living subject and a second light source that isconfigured to emit a beam of near-infrared light to the area ofinterest. The system also includes a handheld probe that is opticallycoupled to the second light source that includes an optical fiberconfigured to deliver the emitted beam of near-infrared light toilluminate the area of interest. The optical fiber is also configured tocollect light that is scattered or light that is emitted from a contrastagent introduced into target tissues in the area of interest in responseto illumination by the second light source. A first imaging device isalso included in the system. The first imaging device is opticallycoupled to the handheld probe and is configured to detect the collectedlight and to generate a corresponding signal that includes collectedlight data. The handheld probe is further configured to transmit thecollected light to the first imaging device, through the optical fiber.The system further includes a second imaging device that is configuredto detect visible light that is emitted from the area of interest inresponse to illumination by the first light source, and to generate acorresponding signal including visible light data. A third imagingdevice is also included in the system, which is configured to detectnear-infrared light having a first predetermined wavelength that isemitted from the area of interest, in response to illumination by thesecond light source, and which is also configured to generate acorresponding signal including a first set of near-infrared light data.In addition, the system includes a fourth imaging device that isconfigured to detect near-infrared light having a second predeterminedwavelength that is different from the first predetermined wavelength andthat is emitted from the area of interest, in response to illuminationby the second light source, and the fourth imaging device is alsoconfigured to generate a corresponding signal that includes a second setof near-infrared light data. A display for displaying at least onevisual representation of data is further included in the system. Also,the system includes a controller that is in communication with each ofthe first light source, second light source, first imaging device,second imaging device, third imaging device, fourth imaging device, anddisplay. The controller may include one or more programmable processorsthat are operative to cause a computer to perform specific functions. Inthis embodiment, the controller is programmed to generate at least onereal-time integrated visual representation of the area of interest fromeach of the collected light data, visible light data, first set ofnear-infrared light data, and second set of near-infrared light data,and to display the real-time visual representation on the display forguidance during the diagnostic or therapeutic procedure.

In one embodiment, the contrast agent includes a Raman probe and/or afluorescence probe and the collected light data includes Raman dataand/or fluorescence data, respectively. In this embodiment, theintegrated visual representation includes a wide-field image of the areaof interest that is generated from the visible light data, a laserexcitation image of a selected area of the area of interest that isdefined within the wide-field image and that is generated from at leastone of the generated first set of near-infrared light data and thegenerated second set of near-infrared light data, and a Raman imagegenerated from the Raman data and/or a fluorescence image generated fromthe fluorescence data. The Raman image and/or fluorescence image isdefined within the wide-field image and the laser excitation image, asan overlay image on the laser excitation image.

In one embodiment, the first imaging device includes a spectrometer andeach of the second imaging device, third imaging device, and fourthimaging device includes a CCD camera.

In another aspect, the present invention relates to an imaging systemusing integrated bright-field imaging, near-infrared imaging, and Ramanimaging and/or fluorescence imaging for intraoperatively evaluatingtarget tissues in an area of interest of a living subject. In oneembodiment, the system includes a first light source for delivering abeam of visible light to the area of interest and a second light sourcefor delivering a beam of near-infrared light to the area of interest.The system also includes a Raman and/or fluorescence imaging means thatincludes a handheld probe optically coupled to the second light source,for delivering the near infrared light to illuminate target tissues ofthe area of interest, and for collecting scattered light and/or emittedlight from a corresponding Raman probe and/or fluorescence probe that isintroduced into the target tissues and illuminated by the second lightsource. The system further includes a first imaging device that is incommunication with the handheld probe, for obtaining Raman data and/orfluorescence data from the collected light. In this embodiment, thefirst imaging device includes a spectrometer. A bright-field imagingmeans is also included in the system according to this embodiment. Thebright-field imaging means includes: an optical port; a system lensincluding a UV-NIR compact lens and a first achromatic correction lens;a silver mirror; a first dichroic mirror and a second dichroic mirror; afirst shortpass filter and a second shortpass filter; a neutral densityfilter; a bandpass filter; a longpass filter; a second achromatic lens,a third achromatic lens, and a fourth achromatic lens; a second imagingdevice for obtaining visible light data from visible light emitted fromthe area of interest in response to illumination by the first lightsource; a third imaging device for obtaining a first set ofnear-infrared data from light having a first predetermined wavelengththat is emitted from the area of interest in response to illumination bythe second light source; and, a fourth imaging device for obtaining asecond set of near infrared data from light having a secondpredetermined wavelength that is different from the first predeterminedwavelength and that is emitted from the area of interest in response toillumination by the second light source. Each of the second imagingdevice, third imaging device, and fourth imaging device include a CCDcamera.

In one embodiment, the optical port and the first imaging device definea first optical path between them that includes the silver mirror, thefirst dichroic mirror, the second dichroic mirror, and the secondachromatic lens, where the optical port and the second imaging devicedefine a second optical path between them that includes the silvermirror, first dichroic mirror, second dichroic mirror, neutral densityfilter, and third achromatic lens. The optical port and the thirdimaging device define a third optical path between them that includesthe silver mirror, first dichroic mirror, longpass filter, bandpassfilter, and fourth achromatic lens. The system according to thisembodiment also includes a display for displaying at least one visualrepresentation of data, and a controller in communication with each ofthe first light source, second light source, first imaging device,second imaging device, third imaging device, fourth imaging device, anddisplay. The controller may include one or more programmable processorsthat are operative to cause a computer to perform specific functions. Inthis embodiment, the controller is programmed for generating in realtime an integrated visual representation of the area of interest fromthe collected light data, first set of near-infrared data, second set ofnear-infrared data, and displaying the integrated visual representationon the display, to provide guidance for performing a diagnostic ortherapeutic procedure.

In one embodiment, the real-time integrated visual representation of thearea of interest includes a wide-field image of the area of interestgenerated from the visible light data, a laser excitation image of apredetermined area defined within the wide-field image that is generatedfrom the first set of near-infrared data and/or the second set ofnear-infrared data, and a Raman image and/or fluorescence image that isdefined within the laser excitation image and that is generated fromcorresponding Raman data and/or fluorescence data. The Raman imageand/or fluorescence image is an overlay image on the laser excitationimage.

In one embodiment, the integrated visual representation of the area ofinterest includes a wide-field image of the area of interest generatedfrom the visible light data, a laser excitation image of a predeterminedarea defined within the wide-field image that is generated from at lestone of the first set of near-infrared data and the second set ofnear-infrared data, and at least one of a Raman image and a fluorescenceimage that is generated from a corresponding at least one of the Ramandata and fluorescence data. The laser excitation image is an overlayimage on the wide-field image and represents the location of thedelivered beam of near-infrared light within the area of interest. TheRaman data and/or fluorescence data is represented by a signal that,when exceeding a predefined threshold level, signifies disease in thetarget tissues.

Further, the Raman image and/or the fluorescence image is a coloroverlay image on the laser excitation image, having an opacityrepresentative of the level of the signal exceeding the predefinedthreshold level, and the opacity of the color overlay image decays overtime to be progressively more translucent relative to the laserexcitation image.

In yet another aspect, the present invention relates to a method forintraoperatively providing anatomical guidance in a diagnostic ortherapeutic procedure. In one embodiment, the method includes the stepsof introducing at least one contrast agent into target tissues in anarea of interest of a living subject, and the step of emitting a beam ofvisible light to the area of interest, using a first light source. Themethod also includes the step of emitting a beam of near-infrared lightto the area of interest, using a second light source, and the step ofdelivering the emitted beam of near-infrared light to illuminate thearea of interest, using an optical fiber of a handheld probe that isoptically coupled to the second light source. In addition, the methodincludes the step of collecting scattered light and/or emitted lightfrom the contrast agent in response to illumination by the second lightsource, using the optical fiber of the handheld probe. The contrastagent includes a Raman probe and/or a fluorescence probe. Further, themethod includes the step of detecting the collected light and generatinga corresponding signal that includes collected light data, using a firstimaging device that is optically coupled to the optical fiber. Theoptical fiber is further configured to deliver the collected light tothe first imaging device. The method also includes the step of detectingvisible light that is emitted from the area of interest in response toillumination by the first light source and generating a correspondingsignal comprising visible light data, using a second imaging device, andthe step of detecting near-infrared light having a first predeterminedwavelength that is emitted from the area of interest in response toillumination by the second light source and generating a correspondingsignal that includes a first set of near-infrared light data, using athird imaging device. Still further, the method includes the step ofdetecting near-infrared light having a second predetermined wavelengththat is different from the first predetermined wavelength and that isemitted from the area of interest in response to illumination by thesecond light source, and generating a corresponding signal that includesa second set of near-infrared light data, using a fourth imaging device,and the step of generating at least one real-time integrated visualrepresentation of the area of interest from the collected light data,visible light data, first set of near-infrared data, and second set ofnear-infrared data, using a controller that is in communication witheach of the first imaging device, second imaging device, third imagingdevice, and fourth imaging device. The controller may include one ormore programmable processors that are operative to cause a computer toperform operational steps according to the method. In this embodiment,the method also includes the step of displaying the real-time integratedvisual representation generated by the controller, for guidance during asurgical procedure, using a display that is in communication with thecontroller.

In one embodiment, the step of generating the real-time integratedvisual representation of the area of interest includes the steps ofgenerating a wide-field image of the area of interest from the visiblelight data, generating a laser excitation image of a selected area ofthe area of interest that is defined within the wide-field image, fromthe first set of near-infrared light data and/or the second set ofnear-infrared light data, and generating a Raman image and/or afluorescence image from the collected light data that is defined withinthe wide-field image and the laser excitation image. The Raman imageand/or fluorescence image is an overlay image on the laser excitationimage.

In one embodiment, the first imaging device includes a spectrometer, andeach of the second imaging device, third imaging device, and fourthimaging device includes a CCD camera.

In yet another aspect, the present invention relates to acomputer-readable medium having stored, computer-executable instructionswhich, when executed by a controller, cause a computer to performspecific functions. In one embodiment, the controller is programmed forcausing a computer to perform functions for intraoperatively providinganatomical guidance in a diagnostic or therapeutic procedure. Thecontroller may include one or more programmable processors. In oneembodiment, the functions include causing a first light source incommunication with the controller to emit a beam of visible light to anarea of interest of a living subject, causing a second light sourceoptically coupled to an optical fiber and in communication with thecontroller to emit a beam of near-infrared light to the area of interestthrough the optical fiber, and causing the optical fiber of the handheldprobe to collect light scattered from a Raman probe and/or light emittedfrom fluorescence probe, in response to illumination by the second lightsource. The Raman probe and/or fluorescence probe is introduced into thetarget tissues in the area of interest. The functions also includecausing a first imaging device that is in communication with thecontroller and the optical fiber to detect the collected light, andcausing the first imaging device to generate a signal from the collectedlight that includes Raman data and/or fluorescence data.

Further, the functions include causing a second imaging device that isin communication with the controller to detect visible light that isemitted from the area of interest in response to illumination by thefirst light source, causing the second imaging device to generate acorresponding signal comprising visible light data, causing a thirdimaging device that is in communication with the controller to detectnear-infrared light having a first predetermined wavelength that isemitted from the area of interest in response to illumination by thesecond light source, and causing the third imaging device to generate acorresponding signal that includes a first set of near-infrared lightdata.

In addition, the functions include causing a fourth imaging device thatis in communication with the controller to detect near-infrared lighthaving a second predetermined wavelength that is different from thefirst predetermined wavelength and that is emitted from the area ofinterest in response to illumination by the second light source, andcausing the fourth imaging device to generate a corresponding signalthat includes a second set of near-infrared light data. Further, thefunctions include generating at least one real-time integrated visualrepresentation of the area of interest from the visible light data,first set of near-infrared data, second set of near-infrared data, andfrom the Raman data and/or fluorescence data, and causing a display incommunication with the controller to display the generated real-timeintegrated visual representation for guidance during a surgicalprocedure.

In one embodiment, the function of generating the real-time integratedvisual representation of the area of interest includes the steps ofgenerating a wide-field image of the area of interest from the visiblelight data, generating a laser excitation image of a selected area ofthe area of interest that is defined within the wide-field image fromthe first set near-infrared light data and/or the second set ofnear-infrared light data, and generating a Raman image from the Ramandata and/or a fluorescence image from the fluorescence data, that isdefined within the wide-field image and the laser excitation image.

In one embodiment, the Raman image and/or fluorescence image is anoverlay image on the laser excitation image. The first imaging deviceincludes a spectrometer, and each of the second imaging device, thirdimaging device, and fourth imaging device includes a CCD camera.

In yet another aspect, the present invention relates to a method forintraoperatively identifying disease in target tissues in an area ofinterest of a living subject, to be resected in a diagnostic ortherapeutic procedure. In one embodiment, the method includes the stepof introducing a Raman probe and/or a fluorescence probe into the areaof interest until the probe has accumulated in the target tissues, thestep of preparing the living subject and the area of interest for asurgical procedure, and the step of initializing an imaging system forintegrated bright-field imaging, near-infrared imaging, and Ramanimaging and/or fluorescence imaging. The method also includes the stepof beginning the diagnostic or therapeutic procedure in the area ofinterest, the step of using a first real-time integrated visualrepresentation of the area of interest and the target tissues that isgenerated by the imaging system to identify a boundary of the targettissues that are diseased, and the step of performing a surgicalresection of the identified diseased target tissues within the boundary.Further, the method includes the steps of, after the surgical resection,using a second displayed real-time integrated visual representation ofthe area of interest and the target tissues, generated by the imagingsystem, to identify any remaining diseased target tissues within theboundary and, if any remaining diseased target tissues are identified,performing a series of further surgical resections on identifiedremaining diseased target tissues corresponding to a respective seriesof real-time integrated visual representations generated by the imagingsystem, until the area of interest is free from diseased target tissues.

In one embodiment, the imaging system includes a first light source thatis configured to emit a beam of visible light to an area of interest ofa living subject and a second light source that is configured to emit abeam of near-infrared light to the area of interest. The system alsoincludes a handheld probe that is optically coupled to the second lightsource, and that includes an optical fiber that is configured to deliverthe emitted beam of near-infrared light to illuminate the area ofinterest and that is also configured to collect light that is scatteredor light that is emitted from a contrast agent introduced into targettissues in the area of interest, in response to illumination by thesecond light source. A first imaging device is also included in thesystem. The first imaging device is optically coupled to the handheldprobe and is configured to detect the collected light and to generate acorresponding signal that includes collected light data. The handheldprobe is further configured to transmit the collected light to the firstimaging device through the optical fiber. The system further includes asecond imaging device that is configured to detect visible light that isemitted from the area of interest in response to illumination by thefirst light source, and to generate a corresponding signal includingvisible light data. A third imaging device is also included in thesystem, which is configured to detect near-infrared light having a firstpredetermined wavelength that is emitted from the area of interest, inresponse to illumination by the second light source, and which is alsoconfigured to generate a corresponding signal including a first set ofnear-infrared light data. In addition, the system includes a fourthimaging device that is configured to detect near-infrared light having asecond predetermined wavelength that is different from the firstpredetermined wavelength, and that is emitted from the area of interestin response to illumination by the second light source. The fourthimaging device is also configured to generate a corresponding signalthat includes a second set of near-infrared light data. A display fordisplaying at least one visual representation of data is furtherincluded in the system. Also, the system includes a controller that isin communication with each of the first light source, second lightsource, first imaging device, second imaging device, third imagingdevice, fourth imaging device, and display. The controller may includeone or more processors that are programmed to cause a computer toperform specific functions. The controller is programmed to generate atleast one real-time integrated visual representation of the area ofinterest from each of the collected light data, visible light data,first set of near-infrared light data, and second set of near-infraredlight data, and to display the at least one real-time visualrepresentation on the display for guidance during the diagnostic ortherapeutic procedure.

In one embodiment of the method, each of the steps of identifyingdiseased target tissues from the displayed visual representationincludes identifying visual representations of the emitted laserexcitation light and visual representations of the collected light datathat are displayed in a selected area of the visual representation.

In one embodiment, the step of identifying the boundary of the targettissues that are diseased and the step of identifying any remainingdiseased target tissues within the boundary includes identifying visualrepresentations of the first set of near-infrared light data, second setof near-infrared light data, and collected light data that are displayedin a selected area of the integrated visual representation. The visualrepresentation of the first set of near-infrared data and second set ofnear-infrared data is a laser excitation image that represents thelocation of the delivered beam of near-infrared light within the area ofinterest, and that is displayed as a color overlay image on thewide-field image.

The signal representing the collected light data that is generated bythe first imaging device, when exceeding a predetermined thresholdlevel, signifies disease in the target tissues. The visualrepresentation of the collected light data is a color overlay image onthe laser excitation image, having an opacity representative of thelevel of the signal exceeding the predefined threshold level. Theopacity of the color overlay image that represents the collected lightdata decays over time to be progressively more translucent relative tothe laser excitation image.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiments, taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application filed contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment, and wherein:

FIG. 1A schematically shows a system for intraoperatively providinganatomical guidance in a diagnostic or therapeutic procedure, accordingto one embodiment of the present invention;

FIG. 1B schematically shows another view of the system according to theembodiment shown in FIG. 1A;

FIG. 2 is a flow chart illustrating operational steps of a method forintraoperatively providing anatomical guidance in a diagnostic ortherapeutic procedure, using the system according to the embodimentshown in FIGS. 1A and 1B, according to one embodiment of the presentinvention;

FIG. 3 schematically shows optical beam paths of a handheldspectroscopic pen device in operation, according to one embodiment ofthe present invention;

FIG. 4 schematically shows a system for wavelength-resolved fluorescenceand Raman measurements, according to one embodiment of the presentinvention;

FIG. 5 illustrates Raman spectra obtained for a standard sample(polystyrene), according to one embodiment of the present invention;

FIG. 6A illustrates fluorescence spectra obtained for variousconcentrations of contrast agents, according to one embodiment of thepresent invention;

FIG. 6B illustrates Raman spectra obtained for various concentrations ofcontrast agents, according to one embodiment of the present invention;

FIG. 7A illustrates fluorescence spectra obtained before backgroundsignal subtraction (upper panel) and after background signal subtraction(lower panel), according to one embodiment of the present invention;

FIG. 7B illustrates Raman spectra obtained before background signalsubtraction (upper panel) and after background signal subtraction (lowerpanel), according to one embodiment of the present invention;

FIG. 8 schematically shows a system for performing tissue penetrationdepth studies of near-infrared fluorescent and SERS contrast agents,according to one embodiment of the present invention;

FIG. 9A illustrates ICG signals as a function of placement depth ofcontrast agents in fresh fat, liver, and lung tissue, according to oneembodiment of the present invention;

FIG. 9B illustrates SERS signals as a function of placement depth ofcontrast agents in fresh fat, liver, and lung tissue, according to oneembodiment of the present invention;

FIG. 10A shows a bright-field image identifying anatomical locations ofa primary tumor and two satellite nodules (dashed circles), according toone embodiment of the present invention;

FIG. 10B shows a bioluminescence image of a mouse, identifying theprimary and satellite tumors (red signals), according to one embodimentof the present invention;

FIG. 11 illustrates ICG signal intensities detected at various locationsidentified in FIGS. 10A and 10B;

FIG. 12A shows a bright-field image identifying a resected tumor (yellowdashed lines) and surgical cavity (cyan dashed line), obtained bydetection of positive and negative tumor margins, with a region having aresidual tumor along the margin of the cavity, as detected by its signalintensity, according to one embodiment of the present invention;

FIG. 12B shows a bioluminescent image identifying a resected tumor(yellow dashed lines) and the surgical cavity (cyan dashed line), wherespectra obtained within the excised tumor are shown in red, those in thesurgical cavity are shown in cyan, and one on the margin of the surgicalcavity is shown by a white arrowhead, according to one embodiment of thepresent invention; and

FIG. 13 illustrates averaged spectra from tumors and positive andnegative margins, according to one embodiment of the present invention.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used.

Certain teens that are used to describe the invention are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the apparatus and methods of theinvention and how to make and use them. For convenience, certain termsmay be highlighted, for example using italics and/or quotation marks.The use of highlighting has no influence on the scope and meaning of aterm; the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification, including examples of any terms discussed herein, isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification. Furthermore,subtitles may be used to help a reader of the specification to readthrough the specification, which the usage of subtitles, however, has noinfluence on the scope of the invention.

OVERVIEW OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. Various embodiments of the invention are now described indetail. Referring to the drawings, like numbers indicate like componentsthroughout the views. As used in the description herein and throughoutthe claims that follow, the meaning of “a”, “an”, and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein and throughout the claims that follow,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise.

The description will be made as to the embodiments of the presentinvention in conjunction with the accompanying drawings in FIGS. 1-13.

Now referring to FIGS. 1A and 1B, in one aspect, the present inventionrelates to a system for intraoperatively providing anatomical guidancein a surgical procedure. In one embodiment, the system includes a firstlight source 100 that is configured to emit a beam of visible light toan area of interest 134 of a living subject, and a second light source102 a that is configured to emit a beam of near-infrared light to thearea of interest 134. The system also includes a handheld probe 104 thatis optically coupled to the second light source 102 a and that includesan optical fiber 106 configured to deliver the emitted beam ofnear-infrared light to illuminate the area of interest 134. The opticalfiber 106 and is also configured to collect light that is scattered 140a and/or or light that is emitted 140 b from a contrast agent 132 a/132b introduced into target tissues in the area of interest 134, inresponse to illumination by the second light source 102 a. A firstimaging device 102 b is also included in the system. The first imagingdevice 102 b is optically coupled to the handheld probe 104 and isconfigured to detect the collected light 140 a/140 b and to generate acorresponding signal that includes collected light data. The handheldprobe 104 is further configured to transmit the collected light 140a/140 b to the first imaging device 102 b through the optical fiber 106.The system further includes a second imaging device 126 that isconfigured to detect visible light 138 that is emitted from the area ofinterest 134 in response to illumination by the first light source 100,and to generate a corresponding signal that includes visible light data.A third imaging device 122 a is also included in the system, which isconfigured to detect near-infrared light 142 a having a firstpredetermined wavelength that is emitted from the area of interest 134,in response to illumination by the second light source 102 a, and whichis also configured to generate a corresponding signal that includes afirst set of near-infrared light data. In addition, the system includesa fourth imaging device 122 b that is configured to detect near-infraredlight 142 b having a second predetermined wavelength that is differentfrom the first predetermined wavelength and that is emitted from thearea of interest 134, in response to illumination by the second lightsource 102 a. The fourth imaging device 122 b is also configured togenerate a corresponding signal that includes a second set ofnear-infrared light data. A display 144 for displaying at least onevisual representation of data is further included in the system. Also,the system includes a controller 130 that is in communication with eachof the first light source 100, second light source 102 a, first imagingdevice 102 b, second imaging device 126, third imaging device 122 a,fourth imaging device 122 b, and display 144. The controller 130 mayinclude one or more programmable processors that are operative to causea computer to perform specific functions. The controller 130 isprogrammed to generate at least one real-time integrated visualrepresentation 146 of the area of interest 134 from each of thecollected light data, visible light data, first set of near-infraredlight data, and second set of near-infrared light data, and to displaythe visual representation on the display 144 for guidance during thesurgical procedure.

In one embodiment, the contrast agent 132 a/132 b includes a Raman probe132 a and/or a fluorescence probe 132 b and the collected light dataincludes Raman data and/or fluorescence data, respectively. In thisembodiment, the integrated visual representation 146 includes awide-field image 146 d of the area of interest 134 that is generatedfrom the visible light data, and a laser excitation image 146 a of aselected area of the area of interest 134 that is defined within thewide-field image 146 d. The laser excitation image 146 a and that isgenerated from at least one of the generated first set of near-infraredlight data and the generated second set of near-infrared light data, andfrom a Raman image 146 b generated from the Raman data and/or afluorescence image 146 c generated from the fluorescence data. The Ramanimage 146 b and/or fluorescence image 146 c is defined within thewide-field image 146 d and the laser excitation image 146 a, as anoverlay image on the laser excitation image 146 a.

In one embodiment, the first imaging device 102 b includes aspectrometer and each of the second imaging device 126, third imagingdevice 122 a, and fourth imaging device 122 b includes a CCD camera.

In another aspect, the present invention relates to an imaging systemusing integrated bright-field imaging, near-infrared imaging, and Ramanimaging and/or fluorescence imaging, for intraoperatively evaluatingtarget tissues in an area of interest 134 of a living subject. In oneembodiment, the system includes a first light source 100 for deliveringa beam of visible light to the area of interest 134 and a second lightsource 102 a for delivering a beam of near-infrared light to the area ofinterest 134. The system also includes a Raman imaging means and/orfluorescence imaging means that includes a handheld probe 104 opticallycoupled to the second light source 102 a, for delivering the nearinfrared light to illuminate target tissues of the area of interest 134,and for collecting scattered light 140 a and/or emitted light 140 b froma corresponding Raman probe 132 a and/or fluorescence probe 132 b thatis introduced into the target tissues and illuminated by the secondlight source 102 a. The system further includes a first imaging device102 b that is in communication with the handheld probe 104, forobtaining Raman data and/or fluorescence data from the collected light140 a/140 b. In this embodiment, the first imaging device 102 b includesa spectrometer.

A bright-field imaging means is also included in the system according tothis embodiment. The bright-field imaging means includes: an opticalport 150; a system lens 108/110 a including a UV-NIR compact lens 108and a first achromatic correction lens 110 a; a silver mirror 112; afirst dichroic mirror 114 a and a second dichroic mirror 116 a; a firstshortpass filter 114 b and a second shortpass filter 116 b; a neutraldensity filter 124; a bandpass filter 120; a longpass filter 118; asecond achromatic lens 110 b, a third achromatic lens 110 c, and afourth achromatic lens 110 d; a second imaging device 126 for obtainingvisible light data from visible light 138 emitted from the area ofinterest 134 in response to illumination by the first light source 100;a third imaging device 122 a for obtaining a first set of near-infrareddata from light 142 a having a first predetermined wavelength that isemitted from the area of interest 134 in response to illumination by thesecond light source 102 a; and, a fourth imaging device 122 b forobtaining a second set of near infrared data from light 142 b having asecond predetermined wavelength that is different from the firstpredetermined wavelength and that is emitted from the area of interest134 in response to illumination by the second light source 102 a. Eachof the second imaging device 126, third imaging device 122 a, and fourthimaging device 122 b includes a CCD camera.

In one embodiment, the optical port 150 and the second imaging device102 b define a first optical path between them that includes the silvermirror 112, the first dichroic mirror 114 a, the second dichroic mirror116 a, and the second achromatic lens 110 b. The optical port 150 andthe fourth imaging device 126 define a second optical path between themthat includes the silver mirror 112, first dichroic mirror 114 a, seconddichroic mirror 116 a, neutral density filter 124, and third achromaticlens 110 c. The optical port 150 and the third imaging device 122 adefine a third optical path between them that includes the silver mirror112, first dichroic mirror 114 a, longpass filter 118, bandpass filter120, and fourth achromatic lens 110 d. The system of this embodimentalso includes a display 144 for displaying at least one visualrepresentation 146 of data, and a controller 130 in communication witheach of the first light source 100, second light source 102 a, firstimaging device 102 b, second imaging device 126, third imaging device122 a, fourth imaging device 122 b, and display 144. The controller mayinclude one or more processors operative to cause a computer to performspecific functions. The controller 130 is programmed for generating inreal time an integrated visual representation 146 of the area ofinterest 134 from the collected light data, visible light data, firstset of near-infrared data, and second set of near-infrared data. Thecontroller 130 is also programmed for displaying the integrated visualrepresentation 146 on the display 144, to provide guidance forperforming a surgical procedure.

In one embodiment, the real-time integrated visual representation 146 ofthe area of interest 134 includes a wide-field image 146 d of the areaof interest 134 that is generated from the visible light data, a laserexcitation image 146 a of a predetermined area defined within thewide-field image 146 d that is generated from the first set ofnear-infrared data and/or the second set of near-infrared data, and aRaman image 146 b and/or fluorescence image 146 c that is defined withinthe laser excitation image 146 a and that is generated fromcorresponding Raman data and/or fluorescence data. The Raman image 146 band/or fluorescence image 146 e is an overlay image on the laserexcitation image 146 a.

In yet another aspect, the present invention relates to a method forintraoperatively providing anatomical guidance in a surgical procedure.In one embodiment, the method includes the steps of introducing at leastone contrast agent 132 a/132 b into target tissues in an area ofinterest 134 of a living subject, and the step of emitting a beam ofvisible light to the area of interest 134, using a first light source100. The method also includes the step of emitting a beam ofnear-infrared light to the area of interest 134, using a second lightsource 102 a, and the step of delivering the emitted beam ofnear-infrared light to illuminate the area of interest 134, using anoptical fiber 106 of a handheld probe 104 that is optically coupled tothe second light source 102 a. In addition, the method includes the stepof collecting scattered light 140 a and/or emitted light 140 b from thecontrast agent 132 a/132 b in response to illumination by the secondlight source 102 a, using the optical fiber 106 of the handheld probe104. The contrast agent 132 a/132 b includes a Raman probe 132 a and/orfluorescence probe 132 b. Further, the method includes the step ofdetecting the collected light 140 a/140 b and generating a correspondingsignal that includes collected light data, using a first imaging device102 b optically coupled to the optical fiber 106. The optical fiber 106is further configured to deliver the collected light 140 a/140 b to thefirst imaging device 102 b.

The method also includes the step of detecting visible light 138 that isemitted from the area of interest 134 in response to illumination by thefirst light source 100 and generating a corresponding signal thatincludes visible light data, using a second imaging device 126. Further,the method includes the step of detecting near-infrared light 142 ahaving a first predetermined wavelength that is emitted from the area ofinterest 134 in response to illumination by the second light source 102a and generating a corresponding signal that includes a first set ofnear-infrared light data, using a third imaging device 122 a. Stillfurther, the method includes the step of detecting near-infrared light142 b having a second predetermined wavelength that is different fromthe first predetermined wavelength and that is emitted from the area ofinterest 134 in response to illumination by the second light source, andgenerating a corresponding signal including a second set ofnear-infrared light data, using a fourth imaging device 122 b. Inaddition, the method includes the step of generating at least onereal-time integrated visual representation 146 of the area of interest134 from the collected light data, visible light data, first set ofnear-infrared data, and second set of near-infrared data, using acontroller 130 that is in communication with each of the first imagingdevice 102 b, second imaging device 126, third imaging device 122 a, andfourth imaging device 122 b. The method further includes the step ofdisplaying the real-time integrated visual representation 146 generatedby the controller 130, for guidance during a surgical procedure, using adisplay 144 that is in communication with the controller 130. Thecontroller 130 may include one or more processors that are operative tocause a computer to perform specific functions.

In one embodiment, the step of generating the real-time integratedvisual representation 146 of the area of interest 134 includes the stepsof generating a wide-field image 146 d of the area of interest 134 fromthe visible light data, generating a laser excitation image 146 a of aselected area of the area of interest 134 that is defined within thewide-field image 146 d, from the first set of near-infrared light dataand/or the second set of near-infrared light data, and generating aRaman image 140 a and/or a fluorescence image 140 b from the collectedlight data, that is defined within the wide-field image 146 d and thelaser excitation image 146 a. The Raman image 140 a and/or fluorescenceimage 140 b is an overlay image on the laser excitation image 146 a.

In one embodiment, the first imaging device 102 b includes aspectrometer, and each of the second imaging device 126, third imagingdevice 122 a, and fourth imaging device 122 b includes a CCD camera.

In yet another aspect, the present invention relates to acomputer-readable medium having stored, computer-executable instructionswhich, when executed by a controller 130, cause a computer to performfunctions for intraoperatively providing anatomical guidance in asurgical procedure. The controller may include one or more programmableprocessors. In one embodiment, the functions include causing a firstlight source 100 in communication with the controller 130 to emit a beamof visible light to an area of interest 134 of a living subject, causinga second light source 102 a that is optically coupled to an opticalfiber 106 and in communication with the controller 130 to emit a beam ofnear-infrared light to the area of interest 134 through the opticalfiber 106, and causing the optical fiber 106 of the handheld probe 104to collect light scattered 140 a from a Raman probe and/or light emitted140 b from a fluorescence probe, in response to illumination by thesecond light source 102 a. The Raman probe 132 a and/or fluorescenceprobe 132 b is introduced into the target tissues in the area ofinterest 134. The functions also include causing a first imaging device102 b that is in communication with the controller 130 and the opticalfiber 106 to detect the collected light 140 a/140 b, and causing thefirst imaging device 102 b to generate a signal from the collected light140 a/140 b that includes Raman data and/or fluorescence data. Further,the functions include causing a second imaging device 126 that is incommunication with the controller 130 to detect visible light 138 thatis emitted from the area of interest 134 in response to illumination bythe first light source 100, causing the second imaging device 126 togenerate a corresponding signal comprising visible light data, causing athird imaging device 122 a that is in communication with the controller130 to detect near-infrared light 142 a having a first predeterminedwavelength that is emitted from the area of interest 134 in response toillumination by the second light source 102 a, and causing the thirdimaging device 122 a to generate a corresponding signal that includes afirst set of near-infrared light data.

In addition, the functions include causing a fourth imaging device 122 bthat is in communication with the controller 130 to detect near-infraredlight 142 b having a second predetermined wavelength that is differentfrom the first predetermined wavelength and that is emitted from thearea of interest 134 in response to illumination by the second lightsource 102 a, and causing the fourth imaging device 122 b to generate acorresponding signal that includes a second set of near-infrared lightdata. Also, the functions include generating at least one real-timeintegrated visual representation 146 of the area of interest 134 fromthe visible light data, first set of near-infrared data, second set ofnear-infrared data, and from the Raman data and/or fluorescence data,and causing a display 144 in communication with the controller 130 todisplay 144 the generated real-time integrated visual representation 146for guidance during a surgical procedure.

In one embodiment, the function of generating the real-time integratedvisual representation 146 of the area of interest 134 includes the stepsof generating a wide-field image 146 d of the area of interest 134 fromthe visible light data, generating a laser excitation image 146 a of aselected area of the area of interest 134 that is defined within thewide-field image 146 d from the first set near-infrared light dataand/or the second set of near-infrared light data, and generating aRaman image 146 b from the Raman data and/or a fluorescence image 146 efrom the fluorescence data, that is defined within the wide-field image146 d and the laser excitation image 146 a.

In one embodiment, the Raman image 146 b and/or fluorescence image 146 cis an overlay image on the laser excitation image 146 a. The firstimaging device 102 b includes a spectrometer, and each of the secondimaging device 126, third imaging device 122 a, and fourth imagingdevice 122 b includes a CCD camera.

Now referring also to FIG. 2, in yet another aspect, the presentinvention relates to a method for intraoperatively identifying diseasein target tissues in an area of interest 134 of a living subject, to beresected in a surgical procedure. In one embodiment, the method includesthe steps 201 and 203 of introducing a Raman probe and/or a fluorescenceprobe into the area of interest 134 until the probe has accumulated inthe target tissues, the step 205 of preparing the living subject and thearea of interest 134 for a surgical procedure, and the step 207 ofinitializing an imaging system for integrated bright-field imaging,near-infrared imaging, and Raman imaging and/or fluorescence imaging.The method also includes the step 209 of beginning the surgicalprocedure in the area of interest 134, the step 211 of using a firstreal-time integrated visual representation of the area of interest 134and the target tissues, generated by the imaging system, to identify aboundary of the target tissues that are diseased, and the step 213 ofperforming a surgical resection of the identified diseased targettissues within the boundary. Further, the method includes the step 215of, after the surgical resection, using a second displayed real-timeintegrated visual representation of the area of interest 134 and thetarget tissues, generated by the imaging system, to identify anyremaining diseased target tissues within the boundary and, the step 219of, if any remaining diseased target tissues are identified, performinga series of further surgical resections on identified remaining diseasedtarget tissues corresponding to a respective series of real-timeintegrated visual representations generated by the imaging system, untilthe area of interest 134 is free from diseased target tissues.

In one embodiment, the imaging system includes a first light source 100that is configured to emit a beam of visible light to an area ofinterest 134 of a living subject and a second light source 102 a that isconfigured to emit a beam of near-infrared light to the area of interest134. The system also includes a handheld probe 104 that is opticallycoupled to the second light source 102 a, and that includes an opticalfiber 106 that is configured to deliver the emitted beam ofnear-infrared light to illuminate the area of interest 134. The opticalfiber 106 is also configured to collect light 140 a that is scattered orlight 140 b that is emitted from a contrast agent 132 a/132 b introducedinto target tissues in the area of interest 134, in response toillumination by the second light source 102 a. A first imaging device102 b is also included in the system. The first imaging device 102 b isoptically coupled to the handheld probe 104 and is configured to detectthe collected light 140 a/140 b and to generate a corresponding signalthat includes collected light data. The handheld probe 104 is furtherconfigured to transmit the collected light 140 a/140 b to the firstimaging device 102 b through the optical fiber 106. The system furtherincludes a second imaging device 126 that is configured to detectvisible light 138 that is emitted from the area of interest 134 inresponse to illumination by the first light source 100, and to generatea corresponding signal including visible light data. A third imagingdevice 122 a is also included in the system, which is configured todetect near-infrared light 142 a having a first predetermined wavelengththat is emitted from the area of interest 134 in response toillumination by the second light source 102 a, and which is alsoconfigured to generate a corresponding signal including a first set ofnear-infrared light data. In addition, the system includes a fourthimaging device 122 b that is configured to detect near-infrared light142 b having a second predetermined wavelength that is different fromthe first predetermined wavelength and that is emitted from the area ofinterest 134, in response to illumination by the second light source 102a. The fourth imaging device 122 b is also configured to generate acorresponding signal that includes a second set of near-infrared lightdata.

A display 144 for displaying at least one visual representation 146 ofdata is further included in the system. Also, the system includes acontroller 130 that is in communication with each of the first lightsource 100, second light source 102 a, first imaging device 102 b,second imaging device 126, third imaging device 122 a, fourth imagingdevice 122 b, and display 144. The controller may include one or moreprocessors operative to cause a computer to perform specific functions.The controller 130 is programmed to generate at least one real-timeintegrated visual representation 146 of the area of interest 134 fromeach of the collected light data, visible light data, first set ofnear-infrared light data, and second set of near-infrared light data,and to display the real-time visual representation 146 on the display144 for guidance during the surgical procedure.

In one embodiment, each of the steps of identifying diseased targettissues from the displayed real-time integrated visual representation146 includes identifying visual representations 146 a of the emittedlaser excitation light 142 a/142 b and visual representations 146 b/146c of the collected light data displayed in a selected area of theintegrated visual representation 146.

IMPLEMENTATIONS AND EXAMPLES OF THE INVENTION

Without intent to limit the scope of the invention, exemplary systemsand methods and their related results according to the embodiments ofthe present invention are given below. Note that titles or subtitles maybe used in the examples for convenience of a reader, which in no wayshould limit the scope of the invention. Moreover, certain theories areproposed and disclosed herein; however, in no way they, whether they areright or wrong, should limit the scope of the invention so long as theinvention is practiced according to the invention without regard for anyparticular theory or scheme of action.

Example 1

This Example relates to a handheld spectroscopic pen device utilizingexogenous contrast agents for in vivo and intra-operative cancerdetection. The handheld spectroscopic pen device and near-infraredcontrast agents are used for intra-operative detection of malignanttumors, based on wavelength-resolved measurements of fluorescence andsurface-enhanced Raman scattering (SERS) signals. The handheldspectroscopic pen device utilizes a near-infrared diode laser (emittingat 785 nm) coupled to a compact head unit for light excitation andcollection. This pen-shaped device removes silica Raman peaks from thefiber optics and attenuates the reflected excitation light, allowing forsensitive analysis of both fluorescence and Raman signals. Its overallperformance has been evaluated by using a fluorescent contrast agent(indocyanine green, or ICG) as well as an SERS contrast agent (pegylatedcolloidal gold). Under in vitro conditions, the detection limits areapproximately 2-5×10⁻¹¹ M for the indocyanine dye and 0.5-1×10⁻¹³ M forthe SERS contrast agent. Ex vivo tissue penetration data show attenuatedbut resolvable fluorescence and Raman signals when the contrast agentsare buried 5-10 mm deep in fresh animal tissues. In vivo studies usingmice bearing bioluminescent 4T1 breast tumors further demonstrate thatthe tumor borders can be precisely detected preoperatively andintraoperatively, and that the contrast signals are strongly correlatedwith tumor bioluminescence. After surgery, the handheld spectroscopicpen device permits further evaluation of both positive and negativetumor margins around the surgical cavity, raising new potential forreal-time tumor detection and image-guided surgery.

Previous work [31-33] with fiberoptic devices for fluorescence and Ramanmeasurements has not examined their suitability for measuring exogenouscontrast agents during surgical procedures. In the present disclosureaccording to this Example, an integrated fiberoptic spectroscopic systemis stably aligned and calibrated and is thus well suited for robustsurgical use. One aspect of this design is that a rigid pen-sizedfiber-optic unit can be used by a surgeon as a handheld device to detectsmall tumors and other lesions in real time during surgery. To addressthe issue of tumor heterogeneity, it is demonstrated that thisspectroscopic system can be combined with injected contrast agents forintraoperative cancer detection and tumor margin delineation. As aresult, much higher detection sensitivity and more consistent tumorsignals are achieved than in previous studies that relied on nativefluorescence or normal Raman scattering.

Reagents

Ultrapure water (18.2 MΩ) was used throughout the studies according tothis Example. Indocyanine green (ICG), 3,3′-diethylthiatricarbocyanineiodide (DTTC), 2,2,2 tribromoethanol, tertiary amyl alcohol, and bovineserum albumin (BSA, 98%) were purchased from Sigma-Aldrich (St. Louis,Mo.). Citrate-stabilized gold colloids (60 nm diameter) at aconcentration of 2.6×10¹⁰ particles/mL were obtained from Ted Pella,Inc. (Redding, Calif.). Dulbecco's Modified Eagle's Medium (DMEM) (4.5g/L glucose, 4.00 mM L-glutamine), fetal bovine serum (FBS),antibiotic/antimycotic solution, and phosphate buffered saline (PBS)were purchased from Thermo Scientific HyClone (Logan, Utah). XenoLightRediJect D-luciferin subtrate was purchased from Caliper Life Sciences(Hopkinton, Mass.). All reagents were used as purchased without furtherpurification.

Handheld Spectroscopic Pen Device

A RamanProbe sampling head and connecting fiberoptics were purchasedfrom InPhotonics (Norwood, Mass.). The cylindrical stainless steelsampling head (diameter 1.3 mm, length 10 cm) was integrated with a 5 mtwo-fiber cable, one for laser excitation and the other for lightcollection. The sampling head and fiber cable were coupled via an FCconnector to a spectrometer designed by Delta Nu (Laramie, Wyo.). Thecombined sampling head and spectrometer system has a wavelength range of800-930 nm with 0.6 nm spectral resolution for fluorescence measurement,and a Raman shift range of 200-2000 cm⁻¹ with 8 cm⁻¹ resolution forRaman measurement. Laser excitation was provided by a continuous-wave200 mW diode laser emitting at 785 nm.

The handheld spectroscopic pen device was compared to a standard Ramanspectrometer (Inspector, 785 nm excitation, 120 mW laser power, 0.6 nmresolution) (DeltaNu, Laramie, Wyo.) to check for wavenumber accuracyacross the entire spectral range. A Raman scattering spectra frompolystyrene was acquired over 5 s from both the handheld spectroscopicpen device and the commercial Raman spectrometer to determine thespectral accuracy of the handheld device. The sensitivity of thehandheld spectroscopic pen device to detect ICG and SERS contrast agentswas also determined. ICG was diluted in BSA solution to concentrationsranging from 25 nM to 50 pM. SERS nanoparticles were diluted in Milli-Qwater to a concentration of 0.2-37.6 pM. Nanoparticle solutions ofdifferent concentrations were transferred (200 μL) into 96 wellhalf-volume black microplates. The handheld spectroscopic pen device wasfixed 10 mm above and centered over each well of the microplate. Signalcollection times for each concentration ranged from 0.1 to 10 s. Therelationship between the integrated signal intensity and the contrastagent concentration was statically analyzed with a linear regressionmodel including calculated 95% confidence intervals. The statisticalanalyses were performed using Origin 6.1 software.

Nanoparticle Contrast Agents

Stock ICG solution was first dissolved in DMSO, and then diluted inaqueous solution containing the albumin protein (40 mg/mL, similar tothe blood protein concentration). Under this condition, the ICGmolecules quickly bound to albumin molecules, resulting in ICG-albumincomplexes with a hydrodynamic size of 4-6 nm (diameter). The use ofalbumin also prevented ICG aggregation and fluorescence quenching [34].Spectrally encoded and PEG-stabilized SERS nanoparticles were preparedaccording to Qian, Nie, and co-workers [26]. Briefly, aqueousdiethylthiatricarbocyanine (DTTC) solution (4 μM) was added dropwise toa gold nanoparticle solution. The optimal SERS signals were detectedwhen approximately 2×10⁴ DTTC molecules were bound to each 60 nm goldparticle. The particles were stabilized by the addition of a thiol-PEGsolution (10 μM) and then purified by centrifugation.

Tissue Penetration Depth Measurement

Porcine tissues used for ex vivo studies were obtained from the Animaland Dairy Science Department at the University of Georgia (Athens, Ga.).Fluorescence and Raman spectra of porcine fat, liver, and lung werecollected over 5-10 s. These tissues were chosen for both theirrelevance to disease processes and for their optical properties. Todetermine the depth at which the handheld spectroscopic pen device candetect fluorescent dyes or SERS nanoparticles in various organs, an 8mm³ section of the tissue was loaded with 20 μL, of either 650 nM ICG or300 μM SERS nanoparticle solution. Next, thinly sliced sections of thecorresponding tissues were laid on top of the contrast agent-loadedspecimen. After each tissue section was applied, fluorescent or Ramanspectra were collected over 0.1-10 s with the handheld spectroscopic pendevice. A distance of 1 cm was maintained between the handheldspectroscopic pen device tip and the top tissue layer, in order tosimulate the handheld spectroscopic pen device position during surgicaluse. A layer of plastic wrap was placed in between the contrast agentloaded tissue and subsequent tissue layers to prevent diffusion ofcontrast agents into the unlabeled tissue slices. Spectra were scaled asnecessary to correct for different integration times and then integratedto obtain the reported signal intensity.

In Vivo and Intra-Operative Measurements

All in vivo murine studies were performed under an approved protocol bythe Emory University IACUC. The mouse mammary carcinoma cell line 4T1,which stably expresses a firefly luciferase gene, was obtained from Dr.Lily Yang at Emory University (Atlanta, Ga.). 4T1 cells were cultured inDMEM containing 10% FBS and IX antibiotic/antimycotic agent. Prior toinjection into mice, the cells were washed two times with PBS anddiluted in sterile PBS to a final concentration of 2×10⁷ cells/mL.Mammary tumors were inoculated into nude mice by the subcutaneousadministration of 2×10⁶ 4T1 cells into the mouse flank. Once the tumorswere approximately 4 mm in diameter, ICG was administered intravenously(i.v.) via a tail vein at a dose of 357 μg/kg. After 24 h, mice wereanesthetized by intraperitoneal (i.p.) injection of a 2.5% solution oftribromoethanol (350 mg/kg). Tumor-bearing mice undergoingbioluminescence imaging were administered i.p. 100 uL of a luciferinsolution (30 mg/mL). Bioluminescent images were acquired on a KodakIn-Vivo FX Imaging System from Carestream Molecular Imaging (Rochester,N.Y.). Corresponding bright-field images were taken for anatomicalreference of the bioluminescence signal. A series of spectra wereacquired on tumor-bearing mice using the handheld spectroscopic pendevice. First, the position of the handheld spectroscopic pen device wasfixed to about 1-2 cm above the location of the acquisition area on themouse. Spectra were collected in 1 s and were obtained from severallocations, including directly over the center of the tumor and theperitumoral region. After the spectra were acquired, the integratedsignal intensity was calculated. The signal intensity was compared toboth the bright-field anatomical location and the bioluminescencesignal.

Handheld Spectroscopic Pen Device Design and Performance.

The handheld spectroscopic pen device connects a handheld sampling head,via a fiberoptic cable, to a spectrometer that can record fluorescenceand Raman signals. The ability to resolve NIR fluorescent and Ramansignals from background tissue arises from the optical filtering thattakes place in the handheld portion of the device, as illustrated inFIGS. 3 and 4. FIG. 3 schematically shows optical beam paths of ahandheld spectroscopic pen device, with excitation light provided from a785 nm laser diode (200 mW output), and having an excitation fiber(“Ex”), collection fiber (“Coll.”), band-pass filter (“BP”), long passfilter (“LP”), dichroic filter (“D”), and reflective mirror (“M”). Asshown, the laser light is transmitted through the excitation fiber intothe pen. A first lens collimates the excitation light. Wavelengthselectivity is provided by a band-pass filter. Excitation light is thenfocused onto the sample of interest. Backscattered light is collectedthrough the same lens. A dichroic mirror and a long pass filterattenuate Rayleigh scattering by a factor of 10⁸ in the collectionfiber. Thus, only Stokes-shifted light is transmitted to thespectrometer. Silica Raman bands arising from the optical fibers areattenuated by physical filtering in both the excitation and emissionoptical paths. The device's overall performance was evaluated bycomparing the polystyrene Raman spectra obtained with the handheldspectroscopic pen device and a standard Raman spectrometer (see FIG. 5).The results show well matched Raman signals between the twospectrometers and also with the literature spectra of polystyrene [35].The differences in peak positions (wavenumbers) are less than 0.5%across the entire range of 200-2000 cm⁻¹.

Detection Sensitivity and Dynamic Range

As depicted in FIG. 4, the handheld spectroscopic pen device allows forsensitive detection of both fluorescent and SERS contrast agents. Alinear relationship is found between the recorded signal intensity andcontrast agent concentration. FIGS. 6A and 6B show the linear regressionmodel fit to the integrated intensity versus concentration curves. Thelinear regression model is shown as a blue line with 95% confidenceintervals shown as dashed red lines. R² is the fit coefficient of thelinear regression model, and has a value of 1 for perfect fits. TheP-values indicate that the slopes of the linear regression aresignificantly different than zero. Further examination shows a narrow95% CI band (red dashed lines) indicating that the regression fit isvery close to the “true” fit for both ICG and SERS contrast agents. Theminimum spectrally resolvable concentrations (that is, limits ofdetection) are 2-5×10⁻¹¹ M for ICG and 0.5-1×10⁻¹³ M for the SERS agent.The Raman reporter dye (diethylthiatricarbocyanine) used here is inresonance with the excitation wavelength at 785 nm, so the phenomenonshould be called surface-enhanced resonance Raman scattering (SERRS).Also, the SERRS nanoparticles are 40-50 fold more sensitive than ICGunder the above-mentioned experimental conditions, primarily because ofthe poor optical properties of ICG (less than 2% quantum yield andfluorescence quenching induced by aggregation). The maximum detectableconcentration is determined by detector signal saturation, theanalog-to-digital converter (16 bits, 2¹⁶=65,536), and the dataintegration time. That is, for low contrast signals, the integrationtime should be increased in order to improve the signal-to-noise ratio,whereas for high contrast signals, the integration time should bereduced to avoid detector saturation (which will allow high-speedacquisition of tumor contrast signals). The dynamic range is thendefined by the low and high limits in which the contrast signalintensity is linear with its concentration. For both fluorescence andRaman measurements, the handheld spectroscopic pen device provides a50-60 fold dynamic range. Accordingly, weak tumor-margin signals thatare 50-60 fold lower than the central tumor signals can be measuredsimultaneously without adjusting the data acquisition parameters, asfurther discussed below.

Spectral Discrimination and Tissue Penetration Depth

An objective of intraoperative use of the handheld spectroscopic pendevice is detection of tumor foci at the margins of the tumor mass,thereby minimizing the risk of positive margins. In practice, areal-time detection system according to aspects of the exemplaryembodiment disclosed in this Example allows the surgeon to remove tumortissue that might have gone undetected, saving the patient from repeatedsurgery and potentially improving survival. Sensitive tumor detection isbased on the use of albumin-bound ICG or SERS nanoparticles as contrastagents. As discussed in more detail later, the main mechanism isbelieved to be “passive tumor targeting” in which nanoparticles areaccumulated and retained in the tumor interstitial space mainly throughthe enhanced permeability and retention (EPR) effect [36, 37].

The ability of the handheld spectroscopic pen device to differentiatecontrast agent signals from the autofluorescence and Raman scattering ofmajor tissue/organ types (i.e. fat, liver and lung) was first examined.FIG. 4A shows representative spectra of pure ICG, animal fat, and amixture of ICG and animal fat (ICG in fat). At 785 nm excitation, ICGhas a fluorescence peak at 816 nm, while fat has a backgroundfluorescence peak at 805 nm plus resolvable Raman signals at 862, 1070,1297, 1439, and 1652 cm⁻¹ (corresponding to 842, 857, 874, 885, and 902nm in wavelength, respectively). ICG buried in fat has identifiablecontributions of both ICG and fat (e.g., ICG fluorescence at 816 nm andthe fat Raman peaks at 874 and 885 nm).

FIG. 7A illustrates fluorescence spectra of pure ICG, animal fat, and amixture of ICG and animal fat before background subtraction (upperpanel) and after background subtraction (lower panel). FIG. 7Billustrates Raman spectra of pure SERS nanoparticles, animal fat, and amixture of SERS nanoparticles and animal fat before backgroundsubtraction (upper panel) and after background subtraction (lowerpanel). All spectra were taken with the handheld spectroscopic pendevice positioned 1 cm above the top layer of tissue. Spectra wereacquired over 0.1-10 s. The background was obtained by averaging fourdifferent spectra obtained from control tissues, and was subtracted fromthe contrast-enhanced spectra or from single background measurements.Signal intensities relative to that of pure ICG or SERS samples areindicated by scaling factors. The Raman reporter dye wasdiethylthiatricarbocyanine (DTTC);

As shown in FIG. 7A (lower panel), the background signal of fat can beaccurately subtracted, allowing nearly pure ICG contrast signals.Similarly, the data in FIG. 7B (upper and lower panels) show that thebackground Raman spectrum can be subtracted to reveal predominantly theSERS contrast signals. As noted earlier, the ability to detect deepersatellite residual tumors adjacent to the primary tumor is important tocomplete tumor resection and improving patient outcome. To simulate thissurgical scenario, the ability of the handheld spectroscopic pen deviceto detect optical contrast agents below the surface of fat, liver, andlung tissues was examined, by placing contrast agent loaded tissuespecimens below 1-2 mm sections of unlabeled tissue (FIG. 8). FIG. 8schematically shows a system for performing tissue penetration depthstudies of near-infrared fluorescent and SERS contrast agents.

FIGS. 9A and 9B show the relationship between signal intensity and thedepth of ICG or SERS agents deeply placed in ex vivo tissues. Asexpected from light scattering, the contrast signal intensity decreasedalmost exponentially with tissue thickness. ICG can be detected moredeeply in fat than other tissues because fat does not scatter theexcitation light as strongly as lung and liver. This finding haspotentially important applications in lipomatous (fat-rich) tissues suchas breast and some other soft tissues. In addition, lung and liver havemore intense autofluorescence with NIR excitation (likely due toporphyrins and related chromophores in these highly vascularizedorgans), which compromises the ability to distinguish ICG emission fromnative autofluorescence. In comparison, SERS nanoparticles give rise tosharp spectral peaks that are distinct from the broad background,allowing accurate extraction of weak SERS signals under high-attenuationand scattering conditions. Thus, weaker SERS signals can be detected andresolved at a greater tissue depth in comparison with ICG fluorescence.The penetration depth can be further improved by positioning thefiberoptic tip closer to the tissue surface (almost in contact).

In Vivo and Intra-Operative Tumor Detection

In vivo investigations were conducted to test the ability of thehandheld spectroscopic pen device to detect intratumoral deposition ofICG after intravenous infusion. This contrast agent has been approved bythe U.S. Food and Drug Administration (FDA) and is indicated for varioususes in humans, such as for determining cardiac output, hepatic functionand liver blood flow, and for ophthalmic angiography [38]. To assessdegree of tumor contrast enhancement using ICG, mice were used in which4T1 tumor cells (2×10⁶ in number) were subcutaneously injected 18 daysprior to imaging. The tumor cells were genetically engineered to expressthe firefly luciferase gene; intravenous injection of luciferin aftertumor development causes these cells to emit bioluminescent light andallows one to determine the precise location of tumors usingbioluminescence imaging. Thus, ICG contrast enhancement can becorrelated with simultaneous bioluminescence imaging to determinewhether ICG contrast enhancement (if any) originated from tumor sites.On day 17 after tumor cell inoculation, ICG was intravenously infusedinto the mice using a dose of 357 μg/kg, which is the equivalent doseused for human use, and then imaged the mice using the handheldspectroscopic pen device 24 h later. Using bioluminescence imaging, adominant tumor site was identified, along with two satellite tumor sitesalong the track of the needle used for inoculation of tumor cells (FIGS.10A and 10B). A set of 14 spectra was obtained from the mouse using thehandheld spectroscopic pen device.

Specifically, FIG. 10A shows a bright-field image identifying theanatomical locations of a primary 4T1 breast tumor and two satellitenodules (dashed circles). The specific locations for measurement using ahandheld spectroscopic pen device are indicated by numbers 1-12 for theprimary tumor and 13-14 for the satellite nodules. FIG. 10B shows abioluminescence image of the mouse, identifying the primary andsatellite tumors (red signals).

FIG. 11 highlights the high degree of ICG contrast enhancement in thetumors as compared to the surrounding tissues. The intense ICG signalsat locations 5-9, 13, and 14 are indeed correlated with the presence oftumor as determined by bioluminescence. The integrated signalintensities from the tumor areas are nearly 10 times more intense thanthe signals obtained from normal regions. Spectra collected from theadjacent edges (less than 2 mm from the tumor) are still 5-6 timesstronger than that of the more remote areas, providing excellentdelineation of the tumor. After surgical removal of the tumors,bioluminescence imaging shows that the excised tumors are bright and thesurgical cavity is dark (see FIGS. 12A and 12B).

Specifically, FIGS. 12A and 12B show bright-field images (FIG. 12A) andbioluminescent images identifying positive and negative tumor marginsdetected using a handheld spectrometer pen device, including a resectedtumor (yellow dashed lines) and the surgical cavity (cyan dashed line).Spectra obtained within the excised tumor are shown in red, those in thesurgical cavity are shown in cyan, and one on the margin of the surgicalcavity is shown by a white arrowhead. As seen on the bioluminescenceimage, there was a region with residual tumor along the margin of thecavity.

Referring to FIG. 13, spectra recorded by the handheld spectroscopic pendevice indicate 10-fold stronger signals for the excised tumors ascompared to the cavity, which is consistent with the contrast ratio oftumor to healthy tissue found within the living animal (see FIG. 11).

There was a very small area of bioluminescence remaining at the marginof the cavity, corresponding to a positive surgical margin, that was notseen by visual inspection alone. Reexamination of this area with thehandheld spectroscopic pen device revealed an ICG signal that was 5times stronger than for adjacent tissue, again consistent with thecontrast ratios recorded from noninvasive imaging. The ability to obtaina strong ICG signal from tumor, remove the tumor as guided by thehandheld spectroscopic pen device, and obtain real-time pathology aboutthe margin status of both excised tissue and the remaining tumor cavity,are all important features for image-guided surgery.

Results indicate that the observed ICG contrast between tumor and normaltissues is very clear and strong, even though no tumor-targeting ligandsare used in this work. Previous oncology studies utilizing ICG aremainly directed toward sentinel lymph node detection [39-42]. Thesestudies rely on direct intratumoral or peritumoral injections of ICGrather than the intravenous route of administration as used in the studyaccording to the present Example. After intravenous administration, ICGis known to bind to the hydrophobic pockets of serum proteins,especially albumin and lipoproteins [38]. Thus, through protein binding,ICG takes on nanometer scale dimensions, with a hydrodynamic size of 6-8nm diameter. The strong tumor enhancement comes from the enhancedpermeability and retention (EPR) effect [43], in which macromolecules ornanoparticles preferentially accumulate in tumor due to the abnormalneovasculature with large fenestrations and poor lymphatic drainagecharacteristic of tumors. More advanced nanoparticle formulations of ICGhave been reported to facilitate longer circulation of ICG and increasedtumor accumulation for diagnostic and photothermal applications [44-47].Also, targeted contrast agents can be developed by conjugating SERS andother nanoparticles to peptides, monoclonal antibodies, andsmall-molecule ligands for molecular recognition of antigens orreceptors on the surface of tumor cells [48].

In summary, according to the present Example, a handheld spectroscopicdevice was constructed and the use of two near-infrared contrast agentsfor in vivo and intra-operative tumor detection has been shown. Under invitro conditions, the handheld device provides a detection limit of2-5×10⁻¹¹ M for ICG and a detection limit of 0.5-1×10⁻¹³ M for SERS. Thetissue penetration depth is about 5-10 mm depending on the tissue'soptical properties and the ability to resolve weak contrast signals. Inaddition, in vivo studies were carried out by using mouse models bearingbioluminescent 4T1 breast tumors. The results indicate that the tumorborders can be precisely detected preoperatively and intraoperatively,resulting in real-time detection of both positive and negative tumormargins around the surgical cavity. In comparing the two types ofnear-infrared contrast agents, SERS nanoparticles (60-80 nm) providerich spectroscopic information (sharp spectral features), but are muchlarger than the ICG-albumin complexes (4-6 nm). Accordingly, the SERSagent may be better suited for mapping blood vessels and tumorboundaries/peripheries (important for delineating tumor margins),whereas ICG-albumin may be better suited for tumor penetration and rapidclearance.

Example 2

This Example relates to an integrated imaging and spectroscopy systemfor image-guided surgery. According to one embodiment, the system isconfigured to detect the signal from a fluorescent or Raman-active probeintroduced into a patient and localized to a disease area of interest(e.g. a tumor). A surgeon using this system may totally remove adiseased area and verify that the diseased area was successfully andentirely removed.

According to one embodiment of the present Example, a multi-modalimaging system comprises a wide-area imaging system that is configuredfor imaging in the visible and near-infrared light ranges (400-1000 nm),and a narrow-beam combination fiberoptic laser light excitation source(633 nm or 785 nm) and spectroscopy detector. The wide-area imagingsystem has one lens and three cameras: one color camera to detect andrecord visible light (400-610 nm, what a user sees with the unaidedeye); one black and white camera to detect the light from the laserexcitation source (633 nm or 785 nm); and one black and white camera todetect the light emitted from a probe (e.g. 850 nm). Physical opticalfilters (bandpass for emission selectivity, laser line/notch to blocklaser excitation light on all but the “laser camera,” and dichroicmirrors to split the desired light among the three cameras) are used tosplit the light collected from a single lens into the three individualcameras and to provide specificity for the desired wavelengths of lightto reach each camera. The system is used alongside fluorescent (e.g.indocyanine green dye, quantum dot) or surface-enhanced Raman scattering(SERS) probes injected into the subject and accumulated by passive oractive targeting to an area corresponding with diseased tissue. When inuse, the information from the cameras is processed by a computer anddisplayed such that the user may see the visual field; an overlay ontothe image of the visual field shows the position of the laserillumination and the light illumination of the probe (if present). Acomputer uses image processing to enhance the image of the visual field,making it easier to distinguish the position of the probe in relation tothe surrounding tissue. Simultaneously, the fiber-optic laserillumination and spectroscopy detector displays a spectrum of the lightemitted from the area illuminated by the laser light. The spectroscopysystem is operative to detect the fluorescence emission and Raman lightscattering of both native tissue and the introduced probes.

Example 3

This example relates to a method for condensing spectrograph informationrecorded by a “Raman pen” spectrometer onto a wide-field video display(see also the system according to Example 2, above), also referred to as“virtual phosphorescence.” According to one embodiment, the virtualphosphorescence display mode is a way to overlay information recordedcontinuously from a Raman pen spectrometer onto a wide-field image. Datarecorded from the spectrometer is a spectrum (the intensity of light ata given wavelength). For fluorescence probes, data is analyzed in asimple area-under-the-curve (AUC) method (ratio of integratedfluorescence to minimum/background); for Raman scattering probes (andoptionally for fluorescence probes), a computationally more complexdeconvolution method is used (match known spectra to the recordedspectra via optimization). A positive signal is assumed when thefluorescence AUC ratio is over a predetermined threshold or when the AUCratio of the spectra obtained through deconvolution are over apredetermined threshold. In both cases, the predetermined threshold isat least 3 standard deviations above the background signal level, andcorresponds to a significant amount of fluorescent or Raman probe in thesample area of the spectrometer.

When a positive signal is recorded, a false color overlay is placed onthe wide-field image at the location of a laser excitation source whenthe signal was recorded (the location is detected by the cameradedicated for laser tracking). The overlay decays over time. That is,initially the overlay will be bright, but over the course of seconds theoverlay will become progressively more translucent (and so appeardimmer). The decay time is user-selectable, so for very staticconditions, such as when the surgical area is being sweeped by the Ramanpen to locate tumor boundaries, a longer decay time (e.g. 5 seconds) isused to indicate where positive signals are recorded. For dynamicconditions, such as when a surgeon is actively cutting tissue underimage guidance, the decay time is short (e.g. 1 second) to accuratelyindicate where positive tumors are recorded.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toenable others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

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We claim:
 1. A system for intraoperatively providing anatomical guidancein a diagnostic or therapeutic procedure, comprising: (a) a lampconfigured to emit a beam of visible light to an area of interest of aliving subject; (b) a laser configured to emit a beam of near-infraredlight to the area of interest; (c) a probe device configured to be heldby hand optically coupled to the laser but not optically coupled to thelamp, comprising an optical fiber configured to deliver the emitted beamof near-infrared light to illuminate the area of interest and configuredto collect light that is scattered or emitted from a contrast agentintroduced into target tissues in the area of interest, in response toillumination by the laser; (d) a spectrometer optically coupled to theprobe device and configured to detect the collected light and togenerate a corresponding signal that comprises collected light data, andwherein the probe device is further configured to transmit the collectedlight to the spectrometer through the optical fiber of the probe device;(e) a first camera configured to detect visible light that is emittedfrom the area of interest in response to illumination by the lampthrough an optical path that has no overlapping portion with the opticalfiber of the probe device, and to generate a corresponding signalcomprising visible light data; (f) a second camera configured to detectnear-infrared light having a first predetermined wavelength that isemitted from the area of interest in response to illumination by thelaser and to generate a corresponding signal comprising a first set ofnear-infrared light data; (g) a third camera configured to detectnear-infrared light having a second predetermined wavelength that isdifferent from the first predetermined wavelength and that is emittedfrom the area of interest in response to illumination by the laser, andto generate a corresponding signal comprising a second set ofnear-infrared light data; (h) a display for displaying at least onevisual representation of data; and (i) a programmable processor incommunication with each of the lamp, laser, spectrometer, first camera,second camera, third camera, and display, and programmed to generate atleast one real-time integrated visual representation of the area ofinterest from each of the collected light data, visible light data,first set of near-infrared light data, and second set of near-infraredlight data and to display the at least one real-time visualrepresentation on the display, for guidance during the diagnostic ortherapeutic procedure.
 2. The system of claim 1, wherein the contrastagent comprises at least one of a Raman probe agent and a fluorescenceprobe agent and the collected light data comprises at least one of Ramandata and fluorescence data, respectively.
 3. The system of claim 2,wherein the at least one integrated visual representation comprises awide-field image of the area of interest generated from the visiblelight data, a laser excitation image of a selected area of the area ofinterest defined within the wide-field image and generated from at leastone of the generated first set of near-infrared light data and thegenerated second set of near-infrared light data, and at least one of aRaman image generated from the Raman data and a fluorescence imagegenerated from the fluorescence data, wherein the at least one of theRaman image and fluorescence image is defined within the wide-fieldimage and the laser excitation image.
 4. The system of claim 3, whereinthe at least one of the Raman image and the fluorescence image is anoverlay image on the laser excitation image.
 5. The system of claim 1,wherein each of the first, second, and third camera is a CCD camera. 6.An imaging system using integrated bright-field imaging, near-infraredimaging, and at least one of Raman imaging and fluorescence imaging forintraoperatively evaluating target tissues in an area of interest of aliving subject, comprising: (a) a lamp for delivering a beam of visiblelight to the area of interest and a laser for delivering a beam ofnear-infrared light to the area of interest; (b) a Raman andfluorescence imaging device, comprising: (i) a probe device configuredto be held by hand optically coupled to the laser but not opticallycoupled to the lamp, for delivering the near infrared light toilluminate target tissues of the area of interest and for collecting atleast one of scattered light and emitted light from a corresponding atleast one of a Raman probe agent and a fluorescence probe agent that isintroduced into the target tissues and illuminated by the laser; and(ii) a spectrometer in optical communication with the probe device forobtaining at least one of Raman data from the collected scattered lightand fluorescence data from the collected emitted light, respectively,transmitted through the probe device; and (c) a bright-field imagingsystem, comprising: (i) a first camera for obtaining visible light datafrom visible light emitted from the area of interest in response toillumination by the lamp, transmitted through an optical path that hasno overlapping portion with the probe device; (ii) a second camera forobtaining a first set of near-infrared data from light having a firstpredetermined wavelength that is emitted from the area of interest inresponse to illumination by the laser; and (iii) a third camera forobtaining a second set of near infrared data from light having a secondpredetermined wavelength that is different from the first predeterminedwavelength and that is emitted from the area of interest in response toillumination by the laser; further comprising an optical port, a systemlens comprising a UV-NIR compact lens, a first achromatic correctionlens, a first dichroic mirror, a second dichroic mirror, a neutraldensity filter, a bandpass filter, a longpass filter, a secondachromatic lens, a third achromatic lens, and a fourth achromatic lens;wherein the optical port and the first camera define a first opticalpath therebetween having the first dichroic mirror, the second dichroicmirror, and the second achromatic lens; wherein the optical port and thesecond camera define a second optical path therebetween having the firstdichroic mirror, the second dichroic mirror, the neutral density filter,and the third achromatic lens; and wherein the optical port and thethird camera define a third optical path therebetween having the firstdichroic mirror, the longpass filter, the bandpass filter, and thefourth achromatic lens.
 7. The imaging system of claim 6, furthercomprising: (d) a display for displaying at least one visualrepresentation of data; and (e) a programmable processor incommunication with each of the lamp, laser, spectrometer, first camera,second camera, third camera, and display, and programmed for generatingin real time at least one integrated visual representation of the areaof interest from the visible light data, first set of near-infrareddata, second set of near-infrared data, and at least one of the Ramandata and fluorescence data and displaying the integrated visualrepresentation on the display, to provide guidance for performing adiagnostic or therapeutic procedure.
 8. The imaging system of claim 7,wherein the at least one real-time integrated visual representation ofthe area of interest comprises a wide-field image of the area ofinterest generated from the visible light data, a laser excitation imageof a predetermined area defined within the wide-field image that isgenerated from at least one of the first set of near-infrared data andthe second set of near-infrared data, and at least one of a Raman imageand a fluorescence image that is generated from a corresponding at leastone of the Raman data and fluorescence data.
 9. The imaging system ofclaim 8, wherein the laser excitation image is an overlay image on thewide-field image and represents the location of the delivered beam ofnear-infrared light within the area of interest.
 10. The imaging systemof claim 8, wherein the at least one of the Raman data and fluorescencedata is represented by a signal that, when exceeding a predefinedthreshold level, signifies disease in the target tissues.
 11. Theimaging system of claim 10, wherein the at least one of the Raman imageand the fluorescence image is a color overlay image on the laserexcitation image, having an opacity representative of the level of thesignal exceeding the predefined threshold level.
 12. The imaging systemof claim 11, wherein the opacity of the color overlay image decays overtime to be progressively more translucent relative to the laserexcitation image.
 13. The imaging system of claim 6, wherein each of thefirst, second, and third camera is a CCD camera.
 14. A method forintraoperatively providing anatomical guidance in a diagnostic ortherapeutic procedure, comprising the steps of: (a) introducing at leastone contrast agent into target tissues in an area of interest of aliving subject; (b) emitting a beam of visible light to the area ofinterest, using a lamp; (c) emitting a beam of near-infrared light tothe area of interest, using a laser; (d) delivering the emitted beam ofnear-infrared light to illuminate the area of interest, using an opticalfiber of a probe device configured to be held by hand that is opticallycoupled to the laser but not optically coupled to the lamp; (e)collecting at least one of scattered light and emitted light from thecontrast agent in response to illumination by the laser, using theoptical fiber of the probe device, wherein the contrast agent comprisesat least one of a Raman probe agent and a fluorescence probe agent; (f)detecting the collected light and generating a corresponding signal thatcomprises collected light data, using a spectrometer that is opticallycoupled to the optical fiber, and wherein the optical fiber is furtherconfigured to deliver the collected light to the spectrometer throughthe probe device; (g) detecting visible light that is emitted from thearea of interest in response to illumination by the lamp through anoptical path that has no overlapping portion with the optical fiber ofthe probe device, and generating a corresponding signal comprisingvisible light data, using a first camera; (h) detecting near-infraredlight having a first predetermined wavelength that is emitted from thearea of interest in response to illumination by the laser and generatinga corresponding signal comprising a first set of near-infrared lightdata, using a second camera; (i) detecting near-infrared light having asecond predetermined wavelength that is different from the firstpredetermined wavelength and that is emitted from the area of interestin response to illumination by the laser and generating a correspondingsignal comprising a second set of near-infrared light data, using athird camera; (j) generating at least one real-time integrated visualrepresentation of the area of interest from the collected light data,visible light data, first set of near-infrared data, and second set ofnear-infrared data, using a programmable processor in communication witheach of the spectrometer, first camera, second camera, and third camera;and (k) displaying the at least one real-time integrated visualrepresentation generated by the programmable processor, for guidanceduring a diagnostic or therapeutic procedure, using a display incommunication with the programmable processor.
 15. The method of claim14, wherein the step of generating the at least one real-time integratedvisual representation of the area of interest comprises the steps ofgenerating a wide-field image of the area of interest from the visiblelight data, generating a laser excitation image of a selected area ofthe area of interest defined within the wide-field image from at leastone of the first set near-infrared light data and the second set ofnear-infrared light data, and generating at least one of a Raman imageand a fluorescence image, from the collected light data, that is definedwithin the wide-field image and the laser excitation image.
 16. Themethod of claim 14, wherein each of the first, second, and third camerais a CCD camera.
 17. A non-transitory tangible computer-readable mediumhaving stored thereon computer-executable instructions which, whenexecuted by a programmable processor, cause a computer to performfunctions for intraoperatively providing anatomical guidance in asurgical procedure, the functions comprising: (a) causing a lamp incommunication with the programmable processor to emit a beam of visiblelight to an area of interest of a living subject; (b) causing a laseroptically coupled to an optical fiber and in communication with theprogrammable processor to emit a beam of near-infrared light to the areaof interest through the optical fiber, wherein the optical fiber is notoptically coupled to the lamp; (c) causing the optical fiber to collectat least one of light scattered from a Raman probe agent introduced intothe target tissues in response to illumination by the laser and lightemitted from fluorescence probe agent introduced into the target tissuesin response to illumination by the laser; (d) causing a spectrometer incommunication with the programmable processor and the optical fiber todetect at least one of light that is scattered from the Raman probeagent and light that is emitted from the fluorescence probe agent, andcollected through the optical fiber, in response to illumination fromthe second light source laser; (e) causing the spectrometer to generateat least one of a signal from the detected scattered light thatcomprises Raman data and a signal from the detected emitted light thatcomprises fluorescence data, respectively; (f) causing a first camerathat is in communication with the programmable processor to detectvisible light that is emitted from the area of interest in response toillumination by the lamp through an optical path that has no overlappingportion with the optical fiber, and causing the first camera to generatea corresponding signal comprising visible light data; (g) causing asecond camera that is in communication with the programmable processorto detect near-infrared light having a first predetermined wavelengththat is emitted from the area of interest in response to illumination bythe laser and causing the second camera to generate a correspondingsignal comprising a first set of near-infrared light data; (h) causing athird camera that is in communication with the programmable processor todetect near-infrared light having a second predetermined wavelength thatis different from the first predetermined wavelength and that is emittedfrom the area of interest in response to illumination by the laser, andcausing the third camera to generate a corresponding signal comprising asecond set of near-infrared light data; (i) generating at least onereal-time integrated visual representation of the area of interest fromthe visible light data, first set of near-infrared data, second set ofnear-infrared data, and at least one of the Raman data and fluorescencedata; and (j) causing a display in communication with the programmableprocessor to display the generated at least one real-time integratedvisual representation for guidance during a diagnostic or therapeuticprocedure.
 18. The non-transitory tangible computer-readable medium ofclaim 17, wherein the step of generating the at least one real-timeintegrated visual representation of the area of interest comprises thesteps of generating a wide-field image of the area of interest from thevisible light data, generating a laser excitation image of a selectedarea of the area of interest defined within the wide-field image from atleast one of the first set near-infrared light data and the second setof near-infrared light data, and generating at least one of a Ramanimage from the Raman data and a fluorescence image from the fluorescencedata that is defined within the wide-field image and the laserexcitation image.
 19. The non-transitory tangible computer-readablemedium of claim 18, wherein the at least one of the Raman image and thefluorescence image is an overlay image on the laser excitation image.20. The non-transitory tangible computer-readable medium of claim 17,wherein each of the first, second, and third camera is a CCD camera. 21.A method for intraoperatively identifying disease in target tissues inan area of interest of a living subject, to be resected in a diagnosticor therapeutic procedure, comprising the steps of: (a) introducing atleast one of a Raman probe agent and a fluorescence probe agent into thearea of interest until the at least one probe agents has accumulated inthe target tissues; (b) preparing the living subject and the area ofinterest for a diagnostic or therapeutic procedure; (c) initializing animaging system for integrated bright-field imaging, near-infraredimaging, and at least one of Raman imaging and fluorescence imaging; (d)beginning the diagnostic or therapeutic procedure in the area ofinterest; (e) using a first real-time integrated visual representationof the area of interest and the target tissues, generated by the imagingsystem, to identify a boundary of the target tissues that are diseased;(f) performing a surgical resection of the identified diseased targettissues within the boundary; (g) after the surgical resection, using asecond displayed at least one real-time integrated visual representationof the area of interest and the target tissues, generated by the imagingsystem, to identify any remaining diseased target tissues within theboundary; and (h) if any remaining diseased target tissues areidentified, performing a series of further surgical resections onidentified remaining diseased target tissues corresponding to arespective series of real-time integrated visual representationsgenerated by the imaging system, until the area of interest is free fromdiseased target tissues. wherein the imaging system comprises: (i) alamp configured to emit a beam of visible light to an area of interestof a living subject; (ii) a laser configured to emit a beam ofnear-infrared light to the area of interest; (iii) a probe deviceconfigured to be held by hand optically coupled to the laser but notoptically coupled to the lamp, comprising an optical fiber configured todeliver the emitted beam of near-infrared light to illuminate the areaof interest and configured to collect light that is scattered or emittedfrom a contrast agent introduced into target tissues in the area ofinterest, in response to illumination by the laser; (iv) a spectrometeroptically coupled to the probe device and configured to detect thecollected light and to generate a corresponding signal that comprisescollected light data, and wherein the probe device is further configuredto transmit the collected light to the spectrometer through the opticalfiber of the probe device; (v) a first camera configured to detectvisible light that is emitted from the area of interest in response toillumination by the lamp through an optical path that has no overlappingportion with the optical fiber of the probe device, and to generate acorresponding signal comprising visible light data; (vi) a second cameraconfigured to detect near-infrared light having a first predeterminedwavelength that is emitted from the area of interest in response toillumination by the laser and to generate a corresponding signalcomprising a first set of near-infrared light data; (vii) a third cameraconfigured to detect near-infrared light having a second predeterminedwavelength that is different from the first predetermined wavelength andthat is emitted from the area of interest in response to illumination bythe laser, and to generate a corresponding signal comprising a secondset of near-infrared light data; (viii) a display for displaying atleast one visual representation of data; and (ix) a programmableprocessor in communication with each of the lamp, laser, spectrometer,first camera, second camera, third camera, and display, and programmedto generate at least one real-time integrated visual representation ofthe area of interest from each of the collected light data, visiblelight data, first set of near-infrared light data, and second set ofnear-infrared light data and to display the at least one real-timevisual representation on the display, for guidance during the diagnosticor therapeutic procedure.
 22. The method of claim 21, wherein the stepof identifying the boundary of the target tissues that are diseased andthe step of identifying any remaining diseased target tissues within theboundary comprise identifying visual representations of the first set ofnear-infrared light data, second set of near-infrared light data, andcollected light data that are displayed in a selected area of theintegrated visual representation.
 23. The method of claim 21, whereinthe visual representation of the first set of near-infrared data andsecond set of near-infrared data is a laser excitation image thatrepresents the location of the delivered beam of near-infrared lightwithin the area of interest, and that is displayed as a color overlayimage on the wide-field image.
 24. The method of claim 23, wherein thesignal representing the collected light data that is generated byspectrometer, when exceeding a predetermined threshold level, signifiesdisease in the target tissues.
 25. The method of claim 24, wherein thevisual representation of the collected light data is a color overlayimage on the laser excitation image, having an opacity representative ofthe level of the signal exceeding the predefined threshold level. 26.The method of claim 25, wherein the opacity of the color overlay imagethat represents the collected light data decays over time to beprogressively more translucent relative to the laser excitation image.27. The method of claim 21, wherein each of the first, second, and thirdcamera is a CCD camera.